Impact of Quenching Failure of Cy Dyes in Differential Gel Electrophoresis

Background: Differential gel electrophoresis (DIGE) is a technology widely used for protein expression analysis. It is based on labelling with fluorescent Cy dyes. In comparative fluorescence gel electrophoresis experiments, however, unspecific labelling using N-hydroxy-succinimide-ester-based labelling protocols was recently detected. Cross-talk was observed due to failure of the quenching process. Here, the impact of this effect for DIGE experiments was investigated. Methodology/Principal Findings: Experiments to test quenching efficiency were performed in replicate using Escherichia coli lysate. Parameters such as the amount of dye and quencher were varied. Labelling and quenching were reversed in one experiment. Differences in protein spot volumes due to limited quenching were determined. For some spots twice the volume was detected underscoring the importance of proper control of silencing of active dye. Conclusions/Significance: It could be demonstrated that uncontrolled labelling increased protein spot volume, even doubling it in some cases. Moreover, proteins responded differently to the protocol. Such unpredictable and unspecific processes are not acceptable in protein regulation studies so that it is necessary to validate the correct amount of quencher for individual samples before the DIGE experiment is performed. Increase of the concentration of lysine, which is used as quencher, from 10 mM to 2500 mM, was sufficient to silence the dye. Alternatively, active dye molecules can be removed by filtration

Identification of poly(ADP-ribose)polymerase-1 and Ku70/Ku80 as transcriptional regulators of S100A9 gene expression

BACKGROUND: S100 proteins, a multigenic family of non-ubiquitous cytoplasmic Ca(2+)-binding proteins, have been linked to human pathologies in recent years. Dysregulated expression of S100 proteins, including S100A9, has been reported in the epidermis as a response to stress and in association with neoplastic disorders. Recently, we characterized a regulatory element within the S100A9 promotor, referred to as MRE that drives the S100A9 gene expression in a cell type-specific, activation- and differentiation-dependent manner (Kerkhoff et al. (2002) J. Biol. Chem. 277, 41879–41887). RESULTS: In the present study, we investigated transcription factors that bind to MRE. Using the MRE motif for a pull-down assay, poly(ADP-ribose)polymerase-1 (PARP-1) and the heterodimeric complex Ku70/Ku80 were identified by mass spectrometry and confirmed by chromatin immunoprecipitation. Furthermore, TPA-induced S100A9 gene expression in HaCaT keratinocytes was blocked after the pharmacologic inhibition of PARP-1 with 1,5-isoquinolinediol (DiQ). CONCLUSION: The candidates, poly(ADP-ribose)polymerase-1 (PARP-1) and the heterodimeric complex Ku70/Ku80, are known to participate in inflammatory disorders as well as tumorgenesis. The latter may indicate a possible link between S100 and inflammation-associated cancer

Identification of Ocular Autoantigens Associated With Juvenile Idiopathic Arthritis-Associated Uveitis

The purpose of the current study was to analyze the binding patterns of serum autoantibodies from juvenile idiopathic arthritis (JIA) and JIA-associated uveitis (JIAU) patients to proteomes from different ocular tissues and to identify potential ocular autoantigens in JIAU. Proteomes from porcine iris, ciliary body, or retina tissue were isolated, separated using 2D-gel electrophoresis, and transferred to a blotting membrane. The binding pattern of serum antibodies from JIA or JIAU patients or healthy controls to ocular proteins was visualized by using anti-human IgG secondary antibodies and chemiluminescence reaction. Selected protein spots were excised from silver-stained 2D gels and subjected to mass spectrometry. Serum antibodies binding to ocular proteins were detected in all patient groups and healthy controls. Irrespective of the patient groups, serum antibodies bound to 49 different protein spots of the retina proteome, to 53 of the ciliary body proteome, and to 44 of the iris proteome. The relative binding frequency of sera to these iris protein spots was significantly higher in JIAU than in JIA patients or healthy controls. Particularly in JIAU patients, cluster analyses indicated a broad range of serum antibodies directed against ocular antigens, mostly in the iris proteome. Iris proteins frequently bound by serum antibodies in all groups were identified as tubulin beta chain, vimentin, ATP synthase subunit beta, actin, and L-lactate dehydrogenase B chain. Iris proteins exclusively bound by JIAU serum antibodies were heat shock cognate 71 kDa protein and keratin. Although serum autoantibody binding to ocular antigens was not disease-specific, a significant diversity of autoantibodies against a broad range of antigens, particularly from the iris tissue, was detected in JIAU patients. As the iris is a major site of inflammation in JIAU, the present data give further evidence that autoantibodies may be involved in JIAU immunopathology

Preserved Morphology and Physiology of Excitatory Synapses in Profilin1-Deficient Mice

Profilins are important regulators of actin dynamics and have been implicated in activity-dependent morphological changes of dendritic spines and synaptic plasticity. Recently, defective presynaptic excitability and neurotransmitter release of glutamatergic synapses were described for profilin2-deficient mice. Both dendritic spine morphology and synaptic plasticity were fully preserved in these mutants, bringing forward the hypothesis that profilin1 is mainly involved in postsynaptic mechanisms, complementary to the presynaptic role of profilin2. To test the hypothesis and to elucidate the synaptic function of profilin1, we here specifically deleted profilin1 in neurons of the adult forebrain by using conditional knockout mice on a CaMKII-cre-expressing background. Analysis of Golgi-stained hippocampal pyramidal cells and electron micrographs from the CA1 stratum radiatum revealed normal synapse density, spine morphology, and synapse ultrastructure in the absence of profilin1. Moreover, electrophysiological recordings showed that basal synaptic transmission, presynaptic physiology, as well as postsynaptic plasticity were unchanged in profilin1 mutants. Hence, loss of profilin1 had no adverse effects on the morphology and function of excitatory synapses. Our data are in agreement with two different scenarios: i) profilins are not relevant for actin regulation in postsynaptic structures, activity-dependent morphological changes of dendritic spines, and synaptic plasticity or ii) profilin1 and profilin2 have overlapping functions particularly in the postsynaptic compartment. Future analysis of double mutant mice will ultimately unravel whether profilins are relevant for dendritic spine morphology and synaptic plasticity

Quantification with Comparative Fluorescence Gel Electrophoresis (CoFGE)

Comparative two-dimensional fluorescence gel electrophoresis (CoFGE) was developed to enable reproducibility of coordinate assignment for protein spots. To that end, it uses an internal grid formed by a set of pure proteins. The composition of the reference compounds is known, so that, in addition to the use of the marker nodes as anchors for gel warping, their fluorescence signal can serve to estimate the amount of protein present in an analyte gel spot of interest. Here, experimental data are provided to that effect. The reference mixture was distributed in five concentrations across the gel, thus allowing the generation of calibration curves for each marker protein. Quantification was evaluated using replicate separations of Escherichia coli lysate. Expectedly, the method worked best for well-defined proteins spots and could then be performed at less than 20% error. A suitable protein mixture (8-97 kDa) has been composed for this purpose. An abundance of reference slots (40) allows the user to alternate the concentration of the protein marker mix on the gel so that areas of gel distortion can be excluded from the calculation.Die Vergleichende zweidimensionale Fluoreszenz Gelelektrophorese (CoFGE) wurde entwickelt, um eine reproduzierbare Zuordnung der Protein-Spotkoordinaten zu ermöglichen. Dazu wird ein internes Gitter verwendet, das durch eine Mischung reiner Proteine gebildet wird. Da die Zusammensetzung der Referenzsubstanzen bekannt ist, kann ihr Fluoreszenzsignal, zusätzlich zur Verwendung für das Gel-Warping, genutzt werden, um die Menge des im Analytenspot vorhandenen Proteins zu bestimmen. Dazu werden hier experimentelle Daten gezeigt. Die Referenzmischung wurde in fünf Konzentrationen über das Gel verteilt, so dass die Erstellung von Kalibrierkurven für jedes Marker-Protein möglich wurde. Die Quantifizierung wurde an Replikattrennungen von Escherichia coli-Lysat evaluiert. Wie zu erwarten, funktionierte das Verfahren am besten für gut definierte Proteinspots; es konnte dann mit weniger als 20% Fehler durchgeführt werden. Eine geeignete Proteinmischung (8-97 kDa) wurde dafür entwickelt. Die Vielzahl der Referenz-Aufgabepunkte (40) gestattet es dem Nutzer, die Proteinkonzentration der Marker auf dem Gel abzuwechseln, so dass Verzerrungen auf dem Gel von der Berechnung ausgeschlossen werden können

Analysis of CoFGE experiments with Delta2D

Comparative fluorescence gel electrophoresis (CoFGE) was developed to improve coordinate assignment in singular experiments. The method uses an internal reference to both correct for gel-to-gel variation and allow semi-quantitation of gel spots in two-dimensional gel electrophoretic experiments (2D-PAGE). Commercial products have become available which enable users to easily perform these powerful experiments. Software capable of warping gel images is required for data analysis. A manual is provided for the use of Delta2D (DECODON) for this purpose.Die vergleichende Fluoreszenz-Gelelektrophorese (CoFGE) wurde entwickelt, um die Koordinatenzuordnung von Proteinspots in Einzelexperimenten zu verbessern. Die Methode korrigiert die Gel-zu-Gel-Variation mittels eines internen Standards, der gleichzeitig halb-quantitative Konzentrationsaussagen in zweidimensionalen gelelektrophoretischen Trennungen (2D-PAGE) erlaubt. Die Methode wurde inzwischen kommerzialisiert. Für die Datenanalyse ist Software nötig, die Gelverzerrungen ausgleichen kann. Hier wird eine Anleitung für die Anwendung von Delta2D (DECODON) für diesen Zweck bereitgestellt.</p

Albumin aggregation and the re-solubilization of dried serum proteins

Albumin is known for its aggregation. As it is a major component of serum, it may be largely responsible for the difficulties in re-suspending dried serum proteins. We found it necessary to break up the solid pellet using a ball mill and subsequently a chaotropic agent and an organic solvent to generate a homogenous protein solution suitable for mass spectrometry-based protein expression analysis.Albumin ist bekannt für seine Aggregation. Da es eine Hauptkomponente von Serum darstellt, ist es wahrscheinlich hauptsächlich für die Schwierigkeiten bei der Resuspendierung getrockneter Serum-Proteine zuständig. Wir stellten fest, dass es notwendig war, zunächst das trockene Pellet mit einer Kugelmühle zu zerstören. Danach mussten Puffer mit Chaotrop und organischen Lösungsmittel zugefügt werden, um eine homogene Proteinlösung generieren, die für die Massenspektrometrie-basierte Expressionsanalyse geeignet war

Large-Scale Hematopoietic Differentiation of Human Induced Pluripotent Stem Cells Provides Granulocytes or Macrophages for Cell Replacement Therapies

Summary Interleukin-3 (IL-3) is capable of supporting the proliferation of a broad range of hematopoietic cell types, whereas granulocyte colony-stimulating factor (G-CSF) and macrophage CSF (M-CSF) represent critical cytokines in myeloid differentiation. When this was investigated in a pluripotent-stem-cell-based hematopoietic differentiation model, IL-3/G-CSF or IL-3/M-CSF exposure resulted in the continuous generation of myeloid cells from an intermediate myeloid-cell-forming complex containing CD34+ clonogenic progenitor cells for more than 2 months. Whereas IL-3/G-CSF directed differentiation toward CD45+CD11b+CD15+CD16+CD66b+ granulocytic cells of various differentiation stages up to a segmented morphology displaying the capacity of cytokine-directed migration, respiratory burst response, and neutrophil-extracellular-trap formation, exposure to IL-3/M-CSF resulted in CD45+CD11b+CD14+CD163+CD68+ monocyte/macrophage-type cells capable of phagocytosis and cytokine secretion. Hence, we show here that myeloid specification of human pluripotent stem cells by IL-3/G-CSF or IL-3/M-CSF allows for prolonged and large-scale production of myeloid cells, and thus is suited for cell-fate and disease-modeling studies as well as gene- and cell-therapy applications